Fossil fuel is widely recognised as an unsustainable source of petroleum because of depleting supplies and the net contribution of these fuels to the carbon dioxide in the atmosphere. Renewable, carbon neutral fuels are necessary for environmental and economic sustainability. Biofuel derived from oil crops is a potential renewable and carbon neutral alternative to petroleum fuels. Currently, biofuels are produced mainly from soybeans, canola oil, animal fat, palm oil, corn oil and waste cooking oil.
Biofuel from the above sources cannot realistically satisfy even a small fraction of the existing demand for fuels. While researchers are seeking alternative feedstock for biofuel, algae have emerged as one of the most promising sources for biofuel production for three main reasons: (1) the yields of oil from algae are orders of magnitude higher than those for traditional oilseeds; (2) algae can grow in places away from the farmlands and forests, thus minimising the damages caused to ecosystems and food chain systems; and (3) algae can be grown in sewers utilising sewage and next to power-plant smokestacks where they digest pollutants and produce oil.
Algae are phototrophic cell factories, capable of deriving energy from sunlight and carbon from carbon dioxide. Algae convert carbon dioxide to potential biofuels, foods, feeds and high-value bioactives. Algae can provide several different types of renewable biofuels and valuable by-products such as antibiotics.
Not all algae are satisfactory for producing biofuel because of their low oil content or slow growth. Some species of the genus Botryococcus are characterised by an ability to produce high levels of hydrocarbons. For example, Botryococcus braunii is a unique colonial green alga that synthesises and accumulates an unusually high level of hydrocarbons up to 76% dry weight. This alga is a potentially good renewable source of useful lipids, hydrocarbons, polysaccharides, and other specialty chemicals.
The hydrocarbons produced by Botryococcus include (1) n-alkadienes and trienes (Race A), (2) triterpenoid botryococcenes and methylated squalenes (Race B), or (3) a tetraterpenoid, lycopadiene (Race L). Triterpenoid hydrocarbons can be used as feedstock for hydrocracking in an oil refinery to produce octane (gasoline, petrol), kerosene, and diesel, for example. Botryococcenes are preferred over alkadienes and alkatrienes for hydrocracking because botryococcenes will likely be transformed into a biofuel with a higher octane rating. It follows that use of algal lipids or hydrocarbons can greatly reduce the environmental impact associated with using coal and petroleum.
However, the production of photosynthetic fuel oils from B. braunii is not competitive with petroleum derived fuels. One major reason for this is the relatively slow growth rate of B. braunii. Furthermore, the gene(s) that causes the algae to produce botryococcene has not been identified or isolated in the art.
Disclosed in the prior art, however, is a squalene synthase (SEQ ID NO: 10) and an alleged botryococcene synthase (SEQ ID NO: 42), each derived from the Race B B. braunii Berkeley (Showa) strain. The alleged botryococcene synthase was not shown to produce botryococcene by molecular identification of botryococcene. Furthermore, the supporting data are consistent with the alleged botryococcene synthase having squalene synthase activity rather than botryococcene synthase activity as alleged.
Therefore, a need exists for synthetic or recombinant tools to facilitate triterpenoid hydrocarbon, particularly botryococcene, production.